Bestatin-related compounds as immunopotentiator

ABSTRACT

3-Amino-2-hydroxy-4-phenylbutanoic acid and esters thereof as well as new derivatives thereof which are related to bestatin in their chemical structure are active to enhance the immune response in living animals.

SUMMARY OF THE INVENTION

This invention relates to new chemical compounds which are relatedcompound of bestatin and which are useful as immunopotentiator forenhancing the immunological response in animals and humans. Thisinvention also includes the production of these new compounds. Thisinvention further relates to uses of these new compounds and theirrelated known compounds as the immunopotentiator.

BACKGROUND OF THE INVENTION

Bestatin is known to have the immunopotentiating properties (see U.S.Pat. No. 4,029,547 and U.S. Pat. No. 4,189,604). Bestatin is (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucine and has the followingstructure: ##STR1##

We earlier have chemically synthetized some compounds which are relatedto bestatin in their chemical structure and which are represented by thefollowing general formula: ##STR2## wherein R¹ is ##STR3## where R³ ishydrogen, chloro, methyl, nitro, hydroxy or amino and n is 0 or 1, andR² is a (lower)alkyl having 1 to 6 carbon atoms, hydroxy(lower)-alkyl,alkylthioalkyl, carboxamido(lower)alkyl or carboxy(lower)-alkyl,provided that when R¹ is benzyl and R² is isobutyl, the configuration ofthe compound is (2S,3R,2'R), (2S,3S,2'S) or (2S,3S,2'R) (see U.S. Pat.No. 4,189,604 issued on Feb. 19, 1980). The compounds of the formula(II) are the dipeptides which are physiologically active and inhibitaminopeptidase B, leucine aminopeptidase and Bleomycin hydrolase andenhance the anti-tumor effect of bleomycin.

We have further researched bestatin and its related known compounds, andnow we have succeeded to provide further new compounds which are relatedto bestatin in their chemical structure and which have theaminopeptidase-inhibiting activity and the immunopotentiating activity.

An object of this invention is to provide new compounds which exhibitthe immunopotentiating activities. Another object of this invention isto provide processes for the production of these new compounds. Furtherobject is to provide an immunopotentiator comprising these new compoundsor their related known compounds. Other objects and utilities of thisinvention will be clear from the following descriptions.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of this invention, there is provided a newcompound of the formula: ##STR4## wherein R₁ is a hydrogen atom or ahydroxyl group, R₂ is a hydrogen atom or an alkanoyl group of 2-7 carbonatoms or an alkoxycarbonyl group of 2-7 carbon atoms, and R₃ is selectedfrom the groups: ##STR5## where R₄ and R₅ may be the same or differentfrom each other and each are an alkyl group of 1-7 carbon atoms, ahydroxyalkyl group of 1-7 carbon atoms, a mercaptoalkyl group of 1-7carbon atoms, a carboxamidoalkyl group of 2-8 carbon atoms, anaminoalkyl group of 1-7 carbon atoms, a guanidyl-N-alkyl group of 1-7carbon atoms, an alkylmercaptoalkyl group of 2-7 carbon atoms, acarboxyalkyl group of 2-8 carbon atoms, an aryl group, particularlyphenyl, an aralkyl group of 2-8 carbon atoms or a substituted aralkylgroup of 2-8 carbon atoms, n is zero or 1, and the asterisk (*) denotesthe R-configuration or S-configuration.

The new compound of the formula (III) may be in the form of apharmaceutically acceptable acid-addition salt or a pharmaceuticallyacceptable salt (carboxaylate).

According to a preferred embodiment of this invention, there is provideda new compound of the formula: ##STR6## wherein R₁ is a hydrogen atom ora hydroxyl group, R₂ is a hydrogen atom or an alkanoyl group of 2 to 7carbon atoms or an alkoxycarbonyl group of 2 to 7 carbon atoms, and R₆is the group: ##STR7## where R₄ and R₅ are as defined before in respectof the formula (III), and the asterisk (*) each denotes theR-configuration or S-configuration.

Specific examples of the new compound (IIIa) of this invention includethe following:

(1) (2S,3R)-3-amino-2-hydroxy-4-phenyl-1-butanol,

(2)((2S)-2-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol,

(3)(2S)-2-N-[(2S,3'R)-3'-N-acetylamino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol,and

(4)(2S)-2-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl-(S)-leucyl]amino-4-methyl-pentanol.

According to another preferred embodiment of this invention, there isprovided a new compound of the formula: ##STR8## wherein R₁ is ahydrogen atom or a hydroxyl group, R₂ is a hydrogen atom or an alkanoylgroup of 2 to 7 carbon atoms or an alkoxycarbonyl group of 2 to 7 carbonatoms, and R₇ and R₈ may be the same or different from each other andeach are an alkyl group of 1 to 7 carbon atoms, a hydroxyalkyl group of1 to 7 carbon atoms, a mercaptoalkyl group of 1 to 7 carbon atom, anaminoalkyl group of 1 to 7 carbon atoms, a guanidyl-N-alkyl group of 1to 7 carbon atoms, an alkylmercaptoalkyl group, a carboxyalkyl group of2 to 8 carbon atoms, a carboxamidoalkyl group of 2 to 8 carbon atoms, anaryl group, particularly phenyl group, an aralkyl group, particularly aphenyl(C₁ -C₄)alkyl group, especially benzyl, or a substituted aralkylgroup, and n is 0 or 1, and the asterisk (*) denotes the R-configurationor S-configuration.

Specific examples of the new compound of the formula (IIIb) of thisinvention include the following:

(5) (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-leucine,

(6) (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-arginine,

(7) (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(R)-leucine,

(8) (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-glutamicacid, and

(9)(3R)-N-[(2'S,3'R)-3'-amino-2'-phenylbutanoyl-S-leucyl]amino-(2S)-2-hydroxy-4-phenylbutanoicacid.

According to a further preferred embodiment of this invention, there isprovided a new compound of the formula: ##STR9## wherein A is a hydrogenatom or an alkanoyl group of 2 to 5 carbon atoms, especially acetyl andB is the group: ##STR10##

Amongst the new compounds of the general formula (III) according to thisinvention, the new compound of the formula (IIIa) may be prepared by aprocess comprising reducing a compound of the formula (IV): ##STR11##wherein R₁ is a hydrogen atom or a hydroxyl group as defined above, R₂is a hydrogen atom or an alkanoyl group or alkoxycarbonyl group asdefined above, and R₉ is the group: ##STR12## where R₄ and R₅ are eachan alkyl group of 1 to 7 carbon atoms, a hydroxylalkyl group of 1 to 7carbon atoms, a mercaptoalkyl group of 1 to 7 carbon atoms, acarboxaminoalkyl group of 2 to 8 carbon atoms, an aminoalkyl group of 1to 7 carbon atoms, a guanidyl-N-alkyl group of 1 to 7 carbon atoms, analkylmercaptoalkyl group of 2 to 7 carbon atoms, a carboxyalkyl group of2 to 8 carbon atoms, an aryl group, particularly phenyl, an aralkylgroup of 7 to 8 carbon atom, particularly benzyl or a substitutedaralkyl group of 7 to 8 carbon atoms; and R₁₀ is an alkyl group of 1 to7 carbon atoms, especially methyl or ethyl or an aralkyl group of 7 to 8carbon atoms, especially benzyl, and the asterisk (*) denotes theR-configuration or S-configuration, or a protected derivative of thecompound (IV) having protected its functional group which should notparticipate in the reduction, and if necessary, removing the residualprotective group from the resulting reduction product in a known manner.

The starting compound (IV) employed in the above process is usually inthe form of an alkyl or aralkyl (eg. benzyl) ester (carboxylate) of anamino acid, dipeptide or tripeptide which is corresponding to suchcompound that the terminal methylol group (--CH₂ OH) of the finalproduct to be produced is replaced by an alkyl or aralkyl carboxylategroup (--COOR₁₀). When the starting compound (IV) is in the form of analkyl ester of an amino acid, the latter may be, for instance, themethyl ester of(3R)-N-tert-butoxycarbonylamino-(2S)-hydroxy-4-phenylbutanoic acid whichmay be prepared by protecting the amino group of(3R)-amino-(2S)-hydroxy-4-phenylbutanoic acid (Journal of Antibiotics,Vol. 29, No. 5, 600-601 (1976)) with the known amino-protective group,tert-butoxycarbonyl group and then esterifying the protected productwith diazomethane in ethyl ether. When the compound (IV) is in the formof an alkyl ester of a dipeptide, the latter may be, for instance, themethyl ester of(2S,3R)-3-N-tert-butoxycarbonylamino-4-phenylbutanoyl-(S)-leucine whichmay be prepared by protecting the amino group of bestatin (U.S. Pat. No.4,029,547) with tert-butoxycarbonyl group and then esterifying theN-protected bestatin with diazomethane in ethyl ether. When the startingcompound (IV) is in the form of an alkyl ester of a tripeptide, thelatter may be prepared by condensing in a known manner for theconventional systhesis of peptides an alkyl ester of an appropriateamino acid with N-benzyloxycarbonylbestatin which may, in turn, beproduced by reacting bestatin with a benzyloxycarbonylating agent suchas benzyloxycarbonyl-p-nitrophenyl ester, benzyloxycarbonyl azide,benzyloxycarbonyl-N-hydroxysuccinimide ester andbenzyloxycarbonyl-4,6-dimethyl-2-mercaptopyrimidine in aqueous dioxane,tetrahydrofuran, acetonitrile or dimethylformamide.

In order to produce the new compound (IIIa) from the starting compound(IV), the compound (IV) is reduced in a manner known for theconventional conversion of a lower alkyl carboxylate (ester) into thecorresponding alcohol, that is, with a reducing agent which is usuallyused for this purpose, for example, with sodium borohydride in a loweralkanol, either aqueous or anhydrous, at ambient temperature. After thereduction is completed, removal of the remaining amino-protecting groupis carried out by a known deprotecting technique, if necessary. Theamino-protecting benzyloxycarbonyl group may be removed by catalytichydrogenolysis in a known manner in the presence of a palladiumcatalyst, and the amino-protecting tert-butoxycarbonyl group may beremoved by mild acidolysis with hydrogen bromide in acetic acid, withtrifluoroacetic acid or with hydrogen chloride in an organic solvent.

The compound of the formula (IIIa) where R₂ denotes an acyl group, mayalso be produced by acylating in a known manner a corresponding compoundwhere R₂ denotes a hydrogen atom, with an acyl chloride or anhydridewhich has the acyl group to be introduced as the group R₂.

Amongst the new compounds of the formula (III) according to thisinvention, the new compound of the formula (IIIb) which is in the formof a tri-peptide is produced by a process comprising condensing thenaturally occurring bestatin (U.S. Pat. No. 4,029,547), a metabolicproduct of bestatin or an optical isomer thereof represented by theformula (V): ##STR13## wherein R₁, R₂, R₇ and the asterisk (*) are asdefined hereinbefore in respect of the formula (IIIb), with an aminoacid represented by the formula (VI): ##STR14## wherein R₈, n and theasterisk (*) are each as defined hereinbefore in respect of the formula(IIIb) or with a protected derivative of said amino acid (VI) havingprotected its functional group (e.g. amino group, hydroxyl group,carboxyl group, guanidyl group or mercapto group, if exists) whichshould not participate in the condensation, in a manner known for theconventional synthesis of peptides, and if required, removing theresidual protective group(s) from the resulting condensation product togive the desired product (IIIb).

The starting compound of the formula (V) as employed in the process justmentioned above may be prepared by a method as described in the "Journalof Medicinal Chemistry" Vol. 20, 510-515 (1977). The condensation of thestarting compound (V) with the amino acid compound (VI) is performed ata temperature of -20° C. to 25° C. by a known method for synthesis ofpeptides, for example, according to the carbodiimide method usingdicyclohexylcarbodiimide as the condensation agent; according to theactive ester method using e.g. hydroxysuccinimide ester, according tothe active amide method using imidazole and the like; according to theactive azide method using e.g. hydrazine; or according to the mixed acidanhydride method using ethyl chloroformate. The organic solvent in whichthe condensation is conducted may be those employed for the conventionalsynthesis of peptides and includes, for example, ethers such astetrahydrofuran and dioxane; esters such as ethyl acetate; ketones suchas acetone; halogenated hydrocarbons such as methylene chloride; amidessuch as dimethylformamide; and nitriles such as acetonitrile. Theprotected derivative of the amino acid reagent (VI) may preferably be inthe form of the acid-addition salt (for example, p-toluenesulfonate orhydrochloride of a lower alkyl or benzyl ester of the amino acid (VI).When the starting compound (V) is condensed with the amino acid reagent(VI) having blocked its functional group, for example, by thecarbodiimide method, the condensation is achieved preferably in thepresence of an organic tertiary amine such as N-methylmorpholine,triethylamine and the like. The removal of the residual amino-protectinggroup from the condensation product may be done by a deprotectingtechnique known in the chemistry of peptides. For instance, theamino-protecting aralkyloxycarbonyl group, especially benzyl-oxycarbonylgroup may be removed by catalytic hydrogenolysis in the presence of apalladium catalyst, and the alkoxycarbonyl group such astert-butoxycarbonyl group may be removed by mild acidolysis withhydrogen bromide in acetic acid, with trifluoroacetic acid or withhydrogen chloride in an organic solvent such as dioxane, tetrahydrofuranand ethyl acetate.

Further, it is known that 3-amino-2-hydroxy-4-phenylbutanoic acid(abbreviated as AHPA hereinafter) is obtained by hydrolysis of bestatinwith hydrochloric acid (see U.S. Pat. No. 4,189,604) and a lower alkylester and benzyl ester of AHPA, and amide derivatives of AHPA have beenprepared by the present inventors and others.

(2S,3R)-AHPA and its three stereo isomers, namely (2R,3S)-AHPA,(2S,3S)-AHPA and (2R,3R)-AHPA were synthesized by acidolysis of3-N-benzyloxycarbonylamino-2-hydroxy-4-phenylbutyronitrile, followed byseparation procedure as described in U.S. Pat. No. 4,189,604. However,this U.S. patent is silent on the physiological activities of the(2S,3R)-AHPA and the stereo isomers thereof. Only in the "Journal ofMedicinal Chemistry" Vol. 20, No. 4, pp. 510-515 (1977), there isdescribed that (2S,3R)-AHPA exhibits a weak inhibitory activity toaminopeptidase B. In the past, no report has been made about whether(2S,3R)-AHPA and the stereo isomers thereof have the immunopotentiatingactivities, as far as the inventors are aware of.

We have now found that (2S,3R)-AHPA and the stereo isomers thereof aswell as some related known compounds thereof, for example, (2S,3R)-AHPAamide and (2S,3R)-3-amino-2-hydroxy-4-p-hydroxyphenylbutanoic acid(abbreviated as (2S,3R)-p-OH-AHPA) which are represented by the generalformula (VII): ##STR15## wherein R₁ is a hydrogen atom or a hydroxylgroup, and R₁₁ is a hydroxyl group, an alkoxyl group of 1 to 4 carbonatoms, benzyloxy group or an unsubstituted or substituted amino group,exhibits an immunopotentiating activity which is higher than that ofbestatin at a lower dosage.

The alkyl ester and benzyl ester of AHPA as well as the amidederivatives of AHPA may be produced from AHPA by a chemical method asdescribed, for example, in the "Journal of Antibiotics" Vol. 29, No. 5,pp. 600-601 (1976). We have found that when the compound of the formula(VII) is tested by the technique of Delayed Type Hypersensitivity(D.T.H.) using sheep red blood cell as the antigen to be injectedsubcutaneously into footpad of mouse, it even at a low dosage of 0.01mcg to 10 mcg/mouse enhances establishment of the D.T.H. responseagainst sheep red blood cell, and hence that the known compound of theformula (VII) is highly active to enhance the cell-mediated immuneresponse.

In consequence, we have now found that the new compound of the generalformula (I) and the known compound of the general formula (VII) areuseful as the immunopotentiator for enhancing the cell-mediated immuneresponse in living animals including man.

According to a second aspect of this invention, therefore, there isprovided a pharmaceutical composition, useful as immunopotentiator,comprising as active ingredient the compound of the formula (VIII):##STR16## wherein R₁ is a hydrogen atom or a hydroxyl group, R₂ is ahydrogen atom, an alkanoyl group of 2 to 7 carbon atoms or analkoxycarbonyl group of 2 to 7 carbon atoms or an aralkyloxycarbonylgroup of 7 to 8 carbon atoms, especially benzyloxycarbonyl, and D is thegroup: ##STR17## wherein R₄ and R₅ may be the same or different fromeach other and are each an alkyl group of 1 to 7 carbon atoms, ahydroxyalkyl group of 1 to 7 carbon atoms, a mercaptoalkyl group of 1 to7 carbon atoms, a carboxamidoalkyl group of 2 to 8 carbon atoms, anaminoalkyl group of 1 to 7 carbon atoms, a guanidyl-N-alkyl group of 2to 7 carbon atoms, an alkylmercaptoalkyl group of 2 to 7 carbon atoms, acarboxyalkyl group of 2 to 8 carbon atoms, an aryl group, particularlyphenyl, an aralkyl group of 7 to 8 carbon atoms, particularly benzyl, ora substituted aralkyl group of 2 to 8 carbon atoms, n is zero or 1, andthe asterisk (*) denotes the R-configuration or S-configuration, incombination with a pharmaceutically acceptable carrier for activeingredient.

According to a preferred embodiment of the second aspect of thisinvention, there is provided a pharmaceutical composition, useful asimmunopotentiator, comprising as active ingredient the new compound ofthe formula (III) shown hereinbefore.

According to a further preferred embodiment of the second aspect of thisinvention, there is provided a pharmaceutical composition, useful asimmunopotentiator, comprising as active ingredient, a compound of theformula (VIIIa): ##STR18## wherein R₁ is a hydrogen atom or a hydroxylgroup, A is a hydrogen atom or an alkanoyl group of 2 to 5 carbon atoms,especially acetyl, and D' is the group: ##STR19## in combination with apharmaceutically acceptable carrier for the active ingredient.

According to an another aspect of this invention, there is provided aprocess of potentiating the immune response in a living animal includingman, which comprises administering orally, parenterally or intrarectallyinto the animal an immunopotentiatingly effective amount of the compoundof the formula (VIII) or (VIIIa) as described above.

Amongst the new compound of the formula (III), including the newcompounds of the formulae (IIIa) and (IIIb) according to this invention,as well as the known compound of the formula (VII) used in thisinvention, the aforesaid Compound Nos. 1, 2, 3, 4 and 5, as well as theknown compounds (2S,3R)-AHPA, (2R,3R)-AHPA, (2S,3R)-AHPA amide andp-OH-AHPA are soluble in water and advantageously can readily beformulated into injectable aqueous solution according to theconventional pharmaceutical technique, whereas the known compound,bestatin is sparingly soluble in water and cannot be formulated intoinjectable aqueous solution.

In order to estimate the acute toxicity of the compound of the formula(VIII), some of the test compound was interaperitoneally given toICR-strain mice (in groups each consisting of 6 mice, male, 5weeks-aged, average body weight 20 g.), when all the tested micetolerated intraperitoneal administration of (2S,3R)-AHPA;(2S)-2-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenyl-butanoyl]amino-4-methyl-pentanol;(2S)-2-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl-(S)-leucyl]amino-4-methyl-pentanol;(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-leucine; and(2S,3R)-3-amino-2-hydroxy-4-phenyl-butanoyl-(S)-leucyl-(S)-arginine at adosage of 100 mg/kg.

The pharmaceutical composition of this invention, owing to its highimmunopotentiating activity, may widely be utilized in animmunotherapeutic treatment of tumors and also for an agent ofpreventing bacterial infections. The composition of this invention maybe formulated for oral, parenteral or intrarectal administration.Composition in the form of injectable solution may contain 0.1% to 10.0%by weight of the compound (VIII) or (VIIIa) as active ingredient, andalso one or more of a pH-adjuster, buffer, stabilizer, excipient, localanesthetics and an additive for rendering the solution isotonic. Theinjectable solution may be prepared to be adapted for subcutaneous,intramuscular or intravenous injection by any conventionalpharmaceutical technique. Solid composition for oral administrationwhich may be in the form of tablets, coated tablets, granules, powderand capsules, may contain excipients for the active ingredient, and ifrequired, other additives, including disintegrators, lubricants,colorants, flavors and the like. The proportion of the active compoundto the carrier may be at a ratio of 1:1 to 1:100 by weight and mayusually be chosen appropriately depending on the form of the orallyadministrable formulation prepared. Suppository formulations may containexcipients and, if necessary, surfactant and lubricants additionally tothe active compound.

The optimum dosage of the compound (VIII) or (VIIIa) administered will,of course, depend on the mode of administration and the treatment aimed.For men, the unit dosage generally contains from 0.002 mg to 200 mg ofthe compound (VIII) or (VIIIa), which may be administered orally individed doses one or more times per day. The new compound of the formula(IIIa) or (IIIb) used in the composition of this invention may beadministered orally to an adult person at a dosage of 50 mg to 200 mgonce a day, while the known compound of the formula (VI) used in thecomposition of this invention may be administered orally to an adultperson at a dosage of 0.002 mg to 200 mg once a day, by way of aguideline.

The physiological activities of the compounds according to or used bythis invention are tested as follows.

EXAMPLE 1

This Example describes the activity of the compound inhibitory toaminopeptidase B.

The aminopeptidase B-inhibiting activity was determined according to amodification of the method of V. K. Hopsu et al. described in the"Archives of Biochemistry and Biophysics" Vol. 114, 557 (1966). Thus, amixture of 0.8 ml of 0.1 mM of (S)-arginine β-naphthylamide and 1.0 mlof 0.1 M Tris-hydrochloride buffer (pH 7.0) was admixed with 0.7 ml of0.1 M Tris-hydrochloride buffer (pH 7.0) containing the test compoundand then heated at 37° C. for 3 minutes. The enzymatic reaction wasstarted by addition of 0.2 ml of an aminopeptidase B solution which hadbeen purified by the chromatographic method of Hopsu et al. withSephadex G-100 (a product of Pharamcia Fine Chemical Co., Sweden). Afterthe reaction at 37° C. for 30 minutes, the reaction solution was admixedwith 0.6 ml of 1.0 M acetate buffer (pH 4.2) containing 1.0 mg/ml ofdiazonium salt of o-aminoazotoluene (Garnet GBC) and 1.0% of asurfactant "Tween" 20 (a registered trademark) to stop the enzymaticreaction and then allowed to stand at ambient temperature for 15minutes. After this, absorbance (a) at 525 nm of the reaction solutionwas measured by spectrophotometer. For the control, the above procedurewas repeated without the test compound and the adsorbance (b) wasmeasured. Percentages for inhibition to aminopeptidase B were calculatedaccording to the equation. ##EQU1##

Inhibition percentages at various concentrations of the test compoundwere measured, and from the values of inhibition percentages measured50% Inhibition Concentration (IC₅₀) was evaluated. The results aresummarized in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Test No.  Test Compounds IC.sub.50 (mcg/ml)                                   ______________________________________                                        1         Compound No. 1 >50                                                  2         Compound No. 2 0.65                                                 3         Compound No. 3 48.9                                                 4         Compound No. 4 5.4                                                  5         Compound No. 5 0.11                                                 6         Compound No. 6 0.20                                                 7         Compound No. 7 15                                                   8         Compound No. 8 53                                                   9         Bestatin (comparative)                                                                       0.03                                                 ______________________________________                                         Notes:                                                                        Compound No. 1: (2S,3R)3-amino-2-hydroxy-4-phenyl-1-butanol-hydrochloride     (see Example 6 (b))                                                           Compound No. 2:                                                               (2S)2-N--[(2'S,3'R)3amino-2hydroxy-4phenylbutanoyl]amino4-methyl-pentanol     (see Example 7 (b))                                                           Compound No. 3:                                                               (2S)2-N--[(2'S,3'R)3N--acetylamino2hydroxy-4phenylbutanoyl]amino4-methyl-    entanol (see Example 8)                                                        Compound No. 4:                                                               (2S)2-N--[(2'S,3'R)3amino-2hydroxy-4phenylbutanoyl-(S)-leucyl]amino4-meth    l-pentanol (see Example 10 (b))                                                Compound No. 5:                                                               (2S,3R)3-amino-2-hydroxy-4-phenyl-butanoyl-(S)-leucyl-(S)-arginine            (sometimes abbreviated as BST--L--Arg hereinafter, see Example 12 (b))        Compound No. 6:                                                               (2S,3R)3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-leucine              (sometimes abbreviated as BST--L--Leu hereinafter, see Example 11 (b))        Compound No. 7:                                                               (2S,3R)3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(R)-leucine              (sometimes abbreviated as BST--D--Leu hereinafter, see Example 13 (b))        Compound No. 8:                                                               (3R)N--[(2'S,3'R)3amino-2hydroxy-4phenylbutanoyl-(S)-leucyl]amino(2S)-2-h    droxy-4-phenyl-butanoic acid (sometimes abbreviated as BST--AHPA               hereinafter, see Example 15)                                             

EXAMPLE 2

This Example describes the tests for evaluation of theimmunopotentiating activity of the new compound of this invention. Thus,the effect of the new compound on cell-mediated immunity was testedaccording to a known technique of Delayed Type Hypersensitivity (D.T.H.)(see P. H. Lagrange et al. "J. Exp. Med." Vol. 139, 1529-1539 (1974)using mice immunized with sheep red blood cell (SRBC) as antigen whichwas inoculated into the footpad of mice.

A suspension of 10⁸ sheep red blood cells in 0.05 ml of physiologicalsaline were subcutaneously injected at the time of immunization into theright hind footpad of each CDF₁ -mouse under test (female, 8 weeks-aged,5 mice in each group) to establish D.T.H. Simultaneously to theimmunization, to the mouse under test was given orally 0.5 mg/kg of thetest compound as the solution which was prepared by dissolving the testcompound in physiological saline and then filtering the solution by amicropore filter (pore diameter: 0.22 micrometers). Four days later, 10⁸SRBC were subcutaneously injected into the left hind footpad of eachtest mouse for elicitation of the D.T.H. response. 24 Hours after theelicitating injection, the increased thickness of the left hind footpadwas measured with calipers to estimate the degree of swelling in thefootpad. The swelling degree is expressed in term of the valuescalculatec by the equation: ##EQU2## The degree of swelling serves toevaluate the degree of cell-mediated immunity involved. For comparison,a suspension of bestatin dispersed in water was tested in the same wayas above. The test results obtained are summarized in Table 2 below.

                  TABLE 2                                                         ______________________________________                                                                    Degree of                                         Test Compounds                                                                              Dosages (mg/kg)                                                                             swelling (%)                                      ______________________________________                                        Compound No. 1                                                                              0.5           129                                                             0.05          123                                               Compound No. 2                                                                              0.5           126                                                             0.05          106                                               Compound No. 3                                                                              0.5           131                                                             0.05          124                                               Compound No. 4                                                                              0.5           103                                               Compound No. 5                                                                              0.5           135                                               Compound No. 6                                                                              0.5           120                                               Compound No. 7                                                                              0.5           120                                               Compound No. 9                                                                              5             136                                                             0.5           109                                               Bestatin (comparative)                                                                      0.5           140-150                                                         0.05          120-130                                           ______________________________________                                         Note:                                                                         Compound No. 9:                                                               (2S,3R)3-amino-2-hydroxy-4-phenyl-butanoyl-(S)-leucyl-(S)-glutamic acid       (see Example 14).                                                        

EXAMPLE 3

This Example describes the immunopotentiating activity of the(2S,3R)-AHPA, that is, (2S,3R)-amino-2-hydroxy-4-phenylbutanoic acidamongst the known compounds of the formula (VII) used in this invention.

(A) Effect of (2S,3R)-AHPA on cell-mediated immunity

The effect of (2S,3R)-AHPA on cell-mediated immunity was tested in thesame way in Example 2, namely according to the D.T.H. technique usingmice immunized with sheep red blood cell as antigen.

Thus, a suspension of 10⁸ sheep red blood cells in 0.05 ml ofphysiological saline were subcutaneously injected at the time ofimmunization into the right hind footpad of each CDF₁ -mouse under test(female, 8 weeks-aged, 5 mice in each group) to establish D.T.H.Simultaneously to this immunization, to the mouse under test was givenorally 10 mcg/mouse, 1 mcg/mouse or 0.1 mcg/mouse of the test compound,(2S,3R)-AHPA as the aqueous solution which was prepared by dissolvingthe test compound in physiological saline and filtering the solution bya micropore filter (pore diameter: 0.22 micrometers). Four days later,10⁸ SRBC were subcutaneously injected into the left hind footpad of eachtest mouse for elicitation of the D.T.H. response. 24 Hours after theelicitating injection, the increased thickness of the left hind footpadwas measured with calipers to estimate the degree of swelling in thefootpad. The degree of swelling was evaluated. The results obtained arelisted in Table 3 below. By way of comparison, bestatin was tested inthe same way as above. It has been revealed that (2S,3R)-AHPA enhancesthe D.T.H. response much more than bestatin as long as the dosages ofthese compounds are lower than 1 mcg/mouse.

                  TABLE 3                                                         ______________________________________                                        Effect of (2S,3R)-AHPA and bestatin orally administered                       on establishment of D.T.H. response in mice immunized                         with SRBC as antigen                                                                                          Degree                                                          Increase in thick-                                                                          of                                                              ness of footpad                                                                             swelling                                      Test Compounds and dosages                                                                      (×0.1 mm)(±S.D.)                                                                   (%)                                           ______________________________________                                        Control            7.9 ± 1.22                                                                              100                                           (2S,3R)-AHPA 10 mcg/mouse                                                                       10.9 ± 0.57                                                                              138*                                          (2S,3R)-AHPA  1 mcg/mouse                                                                       11.9 ± 0.97                                                                              151*                                          (2S,3R)-AHPA  0.1 mcg/mouse                                                                     11.6 ± 1.85                                                                              147*                                          (2S,3R)-AHPA  0.01 mcg/mouse                                                                    10.6 ± 1.17                                                                              134                                           Bestatin10 mcg/mouse                                                                            12.0 ± 1.25                                                                              152*                                          Bestatin1 mcg/mouse                                                                             10.3 ± 0.96                                                                              130*                                          Bestatin0.1 mcg/mouse                                                                            9.3 ± 2.70                                                                              118                                           ______________________________________                                         *p < 0.05                                                                

(B) Effect of (2S,3R)-AHPA given at low dosage on cell-mediated immunity

The effect of (2S,3R)-AHPA on the D.T.H. response was estimated in thesame manner as in the above procedure (A) of Example 3 by orallyadministering 1 mcg/mouse, 0.1 mcg/mouse or 0.01 mcg/mouse of(2S,3R)-AHPA to each mouse under test. The test results obtained aresummarized in Table 4 below, from which it will be evident that(2S,3R)-AHPA even at a low dosage of 0.01 mcg/mouse is active to enhancethe cell-mediated immunity.

                  TABLE 4                                                         ______________________________________                                        Effect of (2S,3R)-AHPA and bestatin orally administered                       at low dosages on establishment of D.T.H. response in                         mice immunized with SRBC as antigen                                                                           Degree                                                          Increase in thick-                                                                          of                                                              ness of footpad                                                                             swelling                                      Test Compounds and dosages                                                                      (×0.1 mm)(±S.D.)                                                                   (%)                                           ______________________________________                                        Control            8.1 ± 1.13                                                                              100                                           (2S,3R)-AHPA 1 mcg/mouse                                                                        10.8 ± 1.29                                                                              133*                                          (2S,3R)-AHPA 0.1 mcg/mouse                                                                      11.1 ± 0.82                                                                              137*                                          (2S,3R)-AHPA 0.01 mcg/mouse                                                                     10.1 ± 1.52                                                                              125*                                          Bestatin1 mcg/mouse                                                                             10.8 ± 1.08                                                                              133*                                          ______________________________________                                         *p < 0.05                                                                

(C) Effect of mode of administration of (2S,3R)-AHPA on cell-mediatedimmunity

The effect of the mode of administering (2S,3R)-AHPA on the D.T.H.response was estimated in the same manner as in the procedure (A) ofExample 3, except that the test compound (as aqueous solution) wasinjected intraperitoneally in stead of being given orally. The testresults obtained are summarized in Table 5 below.

                  TABLE 5                                                         ______________________________________                                        Effect of (2S,3R)-AHPA orally or intraperitoneally                            administered on establishment of D.T.H. response in                           mice immunized with SRBC as antigen                                                          Degree of swelling (%)                                                          Intraperitoneal                                                                           Oral                                             Test Compounds   injection   administration                                   ______________________________________                                        (2S,3R)-AHPA 10 mcg/mouse                                                                      128*        126*                                             (2S,3R)-AHPA  1 mcg/mouse                                                                      143*        135*                                             Bestatin1 mcg/mouse                                                                            128*        133*                                             (as aqueous                                                                   suspension)                                                                   ______________________________________                                         *p < 0.05                                                                

As shown in Table 5, it will be clear that (2S,3R)-AHPA, either uponintraperitoneal injection or upon oral administration, enhances thecell-mediated immunity as much as or much more than bestatin.

As will be clear from the results of the above Tables 3, 4 and 5, uponoral administration, a dosage of (2S,3R)-AHPA in a range of 0.01 to 1mcg/mouse gives an optimum result in enhancing the D.T.H. response inmice immunized with SRBC as antigen. While, the optimum dosage ofbestatin is in a range of 0.1 to 100 mcg per mouse upon oraladministration, as described in Japanese patent application unexaminedprepublication "Kokai" No. 117435/77.

EXAMPLE 4

Following the procedure (A) of the above Example 3, theimmunopotentiating effect of (2R,3S)-AHPA, (2S,3S)-AHPA and (2R,3R)-AHPAwhich are the stereo isomers of the (2S,3R)-AHPA was estimated by theD.T.H. technique. The test results obtained are shown in Table 6 below.

                  TABLE 6                                                         ______________________________________                                        Effect of (2S,3S)-AHPA, (2R,3S)-AHPA and (2R,3R)-AHPA                         orally given on establishment of D.T.H. response in                           mice immunized with SRBC as antigen                                                                           Degree                                                          Increased in thick-                                                                         of                                                              ness of footpad                                                                             swelling                                      Test Compounds and dosages                                                                      (×0.1 mm)(±S.D.)                                                                   (%)                                           ______________________________________                                        (2S,3S)-AHPA 10 mcg/mouse                                                                       11.9 ± 1.36                                                                              135*                                          (2S,3S)-AHPA  1 mcg/mouse                                                                        0.8 ± 1.56                                                                              111                                           (2R,3S)-AHPA 10 mcg/mouse                                                                       10.6 ± 0.97                                                                              120*                                          (2R,3S)-AHPA  1 mcg/mouse                                                                       11.7 ± 1.15                                                                              133*                                          (2R,3R)-AHPA 10 mcg/mouse                                                                        9.6 ± 0.65                                                                              109                                           (2R,3R)-AHPA  1 mcg/mouse                                                                       11.9 ± 0.74                                                                              135*                                          ______________________________________                                         *p < 0.05                                                                

EXAMPLE 5

This Example describes the effect of an ester and amide of (2S,3R)-AHPAon cell-mediated immunity.

The effect of the various esters of (2S,3R)-AHPA on the D.T.H. responsewas estimated in the same manner as in the procedure (A) of Example 3 byorally administering 10 mcg/mouse or 1 mcg/mouse of (2S,3R)-AHPAmethylester hydrochloride or (2S,3R)-AHPA amide to each mouse undertest. The test results obtained are as listed in Table 7 below, fromwhich it will be evident that the ester of (2S,3R)-AHPA is activesubstantially as much as the free acid form of (2S,3R)-AHPA to enhancethe D.T.H. response and hence the cell-mediated immunity.

                  TABLE 7                                                         ______________________________________                                        Effect of methyl ester or amide of (2S,3R)-AHPA orally                        given on establishment of D.T.H. response in mice                             immunized with SBRC as antigen                                                                                Degree                                                          Increase in thick-                                                                          of                                                              ness of footpad                                                                             swelling                                      Test Compounds and dosages                                                                      (×0.1 mm)(±S.D.)                                                                   (%)                                           ______________________________________                                        Control                8.7 ± 0.65                                                                              100                                       (2S,3R)-AHPA                                                                            10 mcg/mouse                                                                              11.5 ± 1.12                                                                              132*                                      methylester                                                                   hydrochloride                                                                            1 mcg/mouse                                                                              11.7 ± 1.29                                                                              134*                                      (2S,3R)-AHPA                                                                            10 mcg/mouse                                                                              11.5 ± 1.20                                                                              131*                                      amide                                                                                    1 mcg/mouse                                                                               9.8 ± 0.82                                                                              111                                       (2S,3R)-AHPA                                                                            10 mcg/mouse                                                                              11.0 ± 1.05                                                                              126*                                                 1 mcg/mouse                                                                              11.7 ± 1.29                                                                              135*                                      Bestatin   1 mcg/mouse                                                                              11.6 ± 0.91                                                                              133*                                      ______________________________________                                         *p < 0.05                                                                     Notes:                                                                        The (2S,3R)AHPA amide is the compound of formula                              ##STR20##                                                                

EXAMPLE 6

This Example describes the effect of a substituted derivative of(2S,3R)-AHPA on cell-mediated immunity.

The effect of (2S,3R)-3-amino-2-hydroxy-4-p-hydroxyphenylbutanoic acid((2S,3R)-p-OH-AHPA) on the D.T.H. response was estimated in the samemanner as in the procedure (A) of Example 3 by orally administering 10mcg/mouse or 1 mcg/mouse of (2S,3R)-p-OH-AHPA to each mouse under test.The test results obtained are summarized in Table 8 below. From theresults of Table 8, it will be clear that the tested derivative of(2S,3R)-AHPA at the dosages under test enhances likewise thecell-mediated immunity.

                  TABLE 8                                                         ______________________________________                                        Effect of the hydroxylated derivative of (2S,3R)-AHPA                         on establishment of D.T.H. in mice immunized with SRBC                        as antigen                                                                                                    Degree                                                          Increase in thick-                                                                          of                                                              ness of footpad                                                                             swelling                                      Test Compounds and dosages                                                                      (×0.1 mm)(±S.D.)                                                                   (%)                                           ______________________________________                                        (2S,3R)-p-                                                                              10 mcg/mouse                                                                              11.0 ± 1.75                                                                              129*                                      OH--AHPA                                                                                 1 mcg/mouse                                                                               9.7 ± 0.54                                                                              114*                                      Bestatin   1 mcg/mouse                                                                              10.3 ± 1.36                                                                              121*                                      (comparative)                                                                 Control    8.5 ± 0.44                                                                            100                                                     ______________________________________                                         *p < 0.05                                                                

From the results of Examples 3, 4, 5 and 6 as above, it will be clearthat (2S,3R)-AHPA and some derivatives thereof as given at a low dosageare active to enhance significantly the cell-mediated immunity, and alsothat (2S,3R)-AHPA given orally at a dosage of 0.01 to 10 mcg/mouseexhibits the immunopotentiating activity as high as bestatin orallygiven at a dosage of 1 to 10 mcg/mouse, as measured according to theD.T.H. response in mice immunized with SRBC as antigen.

The following Examples 7-16 illustrate the synthetic procedure for theproduction of the new compounds of this invention accordance to theformula (IIIa).

EXAMPLE 7 (a) Synthesis of(2S,3R)-3-N-t-butoxycarbonylamino-2-hydroxy-4-phenyl-1-butanol ##STR21##

N-t-Butoxycarbonyl-(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoic acidmethylester (92.8 mg) was dissolved in 0.44 ml of ethanol, and to theresultant solution was added dropwise at ambient temperature 2.3 ml ofethyl alcohol containing 46.6 mg of sodium borohydride. After this, themixture was stirred for 2 hours at ambient temperature to effect thereduction of the carboxylate group. After the reaction was completed,the reaction mixture was concentrated to dryness under reduced pressureand the solid residue was taken up into 3 ml of ethyl acetate. Thesolution obtained was washed with 2 ml of an aqueous solution of 1%citric acid and then with three 2 ml portions of saturated aqueoussodium chloride. The organic solvent phase in the ethyl acetate wasdried over magnesium sulfate for the drying purpose, and the mixture wasfiltered to remove the magnesium sulfate hydrate therefrom. The filtratewas concentrated to dryness to give 81 mg of a crude solid of the titledcompound. When this was recrystallized from a mixture of 0.5 ml ofethylether and 0.5 ml of n-hexane, there was obtained 68 mg of colorlessneedles. m.p. 113°-115° C. This product gave a value of m/e 282 (M+1) inthe analysis of mass spectrometry.

Elemental analysis-- Found: C, 63.69; H, 8.31; N, 4.70%. Calcd. for C₁₅H₂₃ NO₄ (molecular weight 281.39): C, 64.00; H, 8.26; N, 4.98%.

(b) Synthesis of (2S,3R)-3-amino-2-hydroxy-4-phenyl-1-butanolhydrochloride

The compound (68 mg) obtained in the above step (a) was dissolved in 0.4ml of dry methanol containing 20% hydrogen chloride under ice-coolingand then allowed to stand for 30 minutes to effect removal of thet-butoxycarbonyl group. The reaction mixture was then concentrated todryness and the residue was recrystallized from ethanol-ethylether toafford 36 mg of the titled compound as colorless plate-like crystals.m.p. 123°-125° C. [α]_(D) ²⁵ +28.4° (c 1.0, 1 N-HCl). This product gavea value of m/e 182 in the analysis of mass spectrometry.

Elemental analysis-- Found: C, 54.93; H, 7.46; N, 6.40; Cl, 16.0%.Calcd. for C₁₀ H₁₆ NO₂ Cl (molecular weight 217.72): C, 55.30; H, 7.42;N, 6.44; Cl, 16.3%.

EXAMPLE 8 (a) Synthesis of(2S)-2-N-[(2'S,3'R)-3'-N-t-butoxycarbonylamino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methylpentanol##STR22##

(2S,3R)-3-N-t-Butoxycarbonylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucinemethylester (440 mg) was dissolved in a mixture of 8 ml of ethanol and 8ml of distilled water, and the resulting solution was treated withsodium borohydride in the same manner as in the step (a) of Example 7for the reduction. The crude product so obtained (347 mg) wasrecrystallized from ethyl acetate-ethylether to afford 230 mg of thetitled compound as colorless needles. m.p. 159.5°-160° C. Massspectrometry: m/e 394 (M+1).

Elemental analysis-- Found: C, 63.92; H, 8.76; N, 7.14%. Calcd. for C₂₁H₃₄ N₂ O₅ (molecular weight 394.57): C, 63.92; H, 8.70; N, 7.10%.

(b) Synthesis of(2S)-2-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol

The compound (300 mg) obtained in the above step (a) was dissolved in 3ml of dry methanol containing 20% hydrogen chloride and 1.0% anisoleunder ice-cooling and then allowed to stand for 30 minutes to effectremoval of the t-butoxycarbonyl group. The reaction mixture wasconcentrated to dryness to give 210 mg of the hydrochloride of thetitled compound which was hygrescopic and gave a value of m/e 295 in theanalysis of mass spectrometry. This hydrochloride was taken up in avolume of water and neutralized with aqueous sodium hydrogen carbonate,and the resulting aqueous solution was extracted with n-butanol. Theextract in n-butanol was washed with water and concentrated to drynessto give the free base form of the titled compound as an oil. This oilproduct gave a value of m/e 295 (M+1) in mass spectrometry and itschemical structure was confirmed with reference to the data of IR. andNMR. [α]_(D) ²⁵ -2.5° (c 1.0, 1 N-HCl).

EXAMPLE 9 Synthesis of(2S)-2-N-[(2'S,3'R)-3'-N-acetylamino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol

The compound (42 mg) in the form of the free base obtained in the step(b) of Example 8 as above was dissolved in 0.4 ml of methanol and thesolution obtained was admixed with 0.16 ml of acetic anhydride, followedby allowing to stand at ambient temperature overnight to effectacetylation of the amino group of said compound. The reaction solutionwas concentrated to dryness and the residue was taken up into 2 ml ofn-butanol. The solution in n-butanol was washed with 2 ml of water, thenwith 2 ml of saturated aqueous sodium hydrogen carbonate and finallywith water and subsequently concentrated to dryness to give 32 mg of acrude solid of the titled compound. Recrystallization fromethanol-ethylether gave 20 mg of colorless needles. m.p. 188°-189° C.Mass spectrometry: m/e 336 (M⁺).

Elemental analysis-- Found: C, 64.14; H, 8.35; N, 8.30%. Calcd. for C₁₈H₂₈ N₂ O₄ (molecular weight 336.48): C, 64.25; H, 8.40; N, 8.33%.

EXAMPLE 10 Synthesis of(2S)-2-N-[(2'S,3'R)-3'-N-acetylamino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol

(2S,3R)-3-N-Acetylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucinemethylester (36.4 mg) was reduced with 16 mg of sodium borohydride andthen isolated in the same manner as in the step (a) of Example 8 toafford 20.1 mg of the titled compound. m.p. 187.5°-189° C.

EXAMPLE 11 (a) Synthesis of(2S)-2-N-[(2'S,3'R)-3'-N-benzyloxycarbonylamino-2'-hydroxy-4'-phenylbutanoyl-(S)-leucyl]amino-4-methyl-pentanol##STR23##

(2'S,3'R)-3'-N-Benzyloxycarbonylamino-2'-hydroxy-4'-phenylbutanoyl-(S)-leucyl-(S)-leucinebenzylester (120 mg) was reduced with 38 mg of sodium borohydride andthen isolated in the same manner as in the step (a) of Example 7 to give100 mg of the titled compound as a colorless powder. Mass spectrometry:m/e 528 (M+1).

Elemental ayalysis-- Found: C, 67.98; H, 8.03; N, 5.09%. Calcd. for C₃₀H₄₃ N₂ O₆ (molecular weight 527.75): C, 68.27; H, 8.23; N, 5.31%.

(b) Synthesis of(2S)-2-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl-(S)-leucyl]amino-4-methyl-pentanol

The compound (100 mg) obtained in the step (a) of this Example wasdissolved in a mixture of 1 ml of methanol and 0.3 ml of distilledwater, and the solution was admixed with 20 mg of palladium black as thecatalyst and then subjected to hydrogenolysis at ambient temperaturewith hydrogen gas at 3 atm. overnight. The reaction mixture was filteredto remove the catalyst and the filtrate was concentrated to give asyrup. This syrup was admixed with 2 ml of ethyl acetate and 3 ml of 1 Nhydrochloric acid, and the admixture obtained was extracted with waterto transfer the desired deprotected product into the water. The aqueousextract was concentrated to dryness to give 26 mg of the titled compoundas a colorless powder. m.p. 78°-80° C. Mass spectrometry: m/e 408.

The following Examples 12-16 illustrate the preparation of the newcompounds of this invention according to the formula (IIIb).

EXAMPLE 12 (a) Synthesis of(2S,3R)-3-N-benzyloxycarbonylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-leucinebenzylester ##STR24##

(2S,3R)-3-N-benzyloxycarbonylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucine(442 mg) and (S)-leucine benzylester p-toluenesulfonate (393.3 mg) wereadmixed with 1 ml of tetrahydrofuran, followed by addition of 0.11 ml ofN-methylmorpholine and 115 mg of N-hydroxysuccinimide. The resultingsolution was ice-cooled and then admixed with 227 mg ofdicyclohexylcarbodiimide, followed by stirring for 4 hours underice-cooling. The reaction mixture was filtered to remove the ureaderivative as produced, and the filtrate was concentrated to drynessunder reduced pressure. The solid residue was dissolved in 5 ml of ethylacetate and the solution obtained was washed with 3 ml of 1 Nhydrochloric acid, 3 ml of 1 N aqueous sodium hydroxide and finally with3 ml of distilled water. The organic solvent phase was mixed withanhydrous magnesium sulfate for drying and then the mixture was filteredto remove the magnesium sulfate hydrate. The solution (the filtrate) wasconcentrated to dryness under reduced pressure to give 573 mg of a crudesolid of the titled compound. Crystallization of this product fromacetone-ethylether gave 444 mg of colorless needles. Mass spectrometryof this pure product gave a value of m/e 646 (M+1). The chemicalstructure of the product was confirmed with reference to the data of IR.and NMR.

Elemental analysis-- Found: C, 68.64; H, 6.97; N, 6.30%. Calcd. for C₃₇H₄₇ N₃ O₇ (molecular weight: 645.87): C, 68.80; H, 6.89; N, 6.51%.

(b) Synthesis of(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-leucine

The compound (200 mg) obtained in the above step (a) was taken up into amixture of 10 ml of dioxane and 0.2 ml of distilled water, and theresultant solution was admixed with 60 mg of palladium black as thehydrogenolysis catalyst. The mixture was subjected to the hydrogenolysisat ambient temperature with hydrogen gas at 3 atm. for 48 hours toeffect removal of both the amino-portecting benzyloxycarbonyl group andthe carboxyl-protecting benzyl group. The reaction mixture was filteredto remove the catalyst, and the filtrate was concentrated to drynessunder reduced pressure. Recrystallization of the solid residue frommethanol-ethyl acetate gave 96.8 mg of the titled compound as colorlessneedles. m.p. 216°-219° C. (with foaming). [α]_(D) ²⁵ -37.0° (c 1.0, 1N-HCl). Mass spectrometry: m/e 422 (M+1).

Elemental analysis-- Found: C, 62.89; H, 8.19; N, 9.68%. Calcd. for C₂₂H₃₅ N₃ O₅ (molecular weight: 421.60): C, 62.91; H, 8.39; N, 9.97%.

EXAMPLE 13 (a) Synthesis of(2S,3R)-3-N-benzyloxycarbonylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-N^(G)-nitroarginine benzylester ##STR25##

(2S,3R)-3-N-Benzyloxycarbonylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucine(88.4 mg) and N^(G) -nitroarginine benzylester di-p-toluene-sulfonate(130.8 mg) were dissolved in 1 ml of tetrahydrofuran, followed byaddition of 0.022 ml of N-methylmorpholine and 34 mg ofN-hydroxysuccinimide thereto. The resulting solution was ice-cooled andadmixed with 84 mg of dicyclohexylcarbodiimide, followed by stirring for24 hours under ice-cooling. The reaction mixture was filtered to removetherefrom the urea derivative as produced. The filtrate was concentratedto dryness and the solid residue was dissolved in 5 ml of ethyl acetate.The solution was washed with 5 ml of 1 N hydrochloric acid 5 ml of 1 Naqueous sodium hydroxide and then 5 ml of distilled water andsubsequently mixed with anhydrous magnesium sulfate for drying. Themagnesium sulfate hydrate was filtered off and the filtrate wasconcentrated to dryness to give 142 mg of the titled compound as apowder. Mass spectrometry of this product gave a value of m/e 734 (M+1), and its chemical structure was confirmed with reference to the dataof IR. and NMR.

(b) Synthesis of(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-arginine

The compound (117 mg) obtained in the above step (a) was admixed with amixture of 2.5 ml of dioxane, 0.25 ml of distilled water and 0.25 ml ofglacial acetic acid followed by addition of 25 mg of palladium blackthereto. The mixture was subjected to hydrogenolysis at ambienttemperature with hydrogen gas at 3 atm. for 72 hours, and the reactionmixture was filtered to remove the catalyst. The filtrate wasconcentrated and the concentrated solution was mixed with a small volumeof distilled water and adjusted to pH 2.0 by addition of 6 Nhydrochloric acid. The acidified solution was passed through a column of100 mg of active carbon for chromatography (a product of Wako JunyakuCo., Japan) for adsorption of the desired product. For the purificationpurpose, the carbon column was developed with distilled water and theeluate was concentrated to dryness to give 156 mg of the purifiedproduct as a powder. m.p. 147°-152° C. [α]_(D) ²⁵ -23.5° (c, 1 N-HCl).Mass spectrometry: m/e 465. This product showed a positive reaction toSakaguchi reagent.

EXAMPLE 14 Synthesis of(2S,3)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(R)-leucine

(2S,3R)-3-N-Benzyloxycarbonylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucine(1.11 g) was reacted with 983.3 mg of (R)-leucine benzylesterp-toluenesulfonate and the resulting condensation product was isolatedin the same manner as in the step (a) of Example 12 to give 1.39 g ofthe 3-N-benzyloxycarbonyl-protected derivative of(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(R)-leucine as apowder. This protected product showed a value of m/e 646 in the analysisof mass spectrometry.

This protected product was subjected to the hydrogenolysis with hydrogenover palladium black in the same manner as in the step (b) of Example 12for the deprotection to give a crude powder of the titled compound.Recrystallization of this powder from methanol gave 510 g of colorlessplate-like crystals. m.p. 240°-243° C. Mass spectrometry: m/e 422. Whenthis product was subjected to a silica gel thin layer chromatography ona silica gel plate (Art. 5715, a product of Merck Co., Germany)developed with a mixed solvent of butyl acetate-n-butanol-aceticacid-water (4:4:1:1 by volume), it showed an Rf value of 0.33. While,the isomer product of the Example 12 (b) showed an Rf value of 0.39 inthe same silica gel thin layer chromatography as above.

Elemental analysis--Found: C, 63.10, H, 8.09; N, 9.70%. Calcd. for C₂₂H₃₅ N₃ O₅ : C, 62.91; H, 8.39; N, 9.97%.

EXAMPLE 15 Synthesis of(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-glutamic acid##STR26##

(2S,3R)-3-N-Benzyloxycarbonylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucine(442 mg) was reacted with 500 mg of (S)-glutamic acid benzylesterp-toluenesulfonate and the resulting condensation product was isolatedin the same manner as in the step (a) of Example 12 to give 667 mg ofthe 3-N-benzyloxycarbonyl-protected derivative of(2S,3R)-3-N-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-glutamicacid which gave a value of m/e 662 (M+1) in the analysis of massspectrometry. This protected product (200 mg) was subjected tohydrogenolysis in the same manner as in the step (b) of Example 12 ofthe deprotection to give 73 mg of the titled compound as a colorlesspowder. m.p. 143°-146° C. [α]_(D) ²⁵ -24.7° (c 1.0, 1 N-HCl). Massspectrometry: m/e 438 (M+1).

Elemental analysis--Found C, 57.21, H, 7.31, N, 9.30%. Calcd. for C₂₁H₃₁ N₃ O₇ : C, 57.64, H, 7.16, N, 9.61%.

EXAMPLE 16 Synthesis of (3R)-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl-(S)-leucyl]amino-(2S)-2-hydroxy-4-phenylbutanoicacid ##STR27##

(2S,3R)-3-N-Benzyloxycarbonylamino-2-hydroxy-4-phenylbutanoyl-(S)-leucine(309 mg) was reacted with (2S,3R)-3-amino-2-hydroxy-4-phenylbutyric acidbenzylester hydrochloride (225 mg) and the resulting condensationproduct was isolated in the same manner as in the step (a) of Example 12to give 440 mg of the 3-N-benzyloxycarbonyl-protected derivative of(3R)-N-[(2'S,3'R)-B 3'-amino-2'-hydroxy-4-phenylbutanoyl-(S)-leucyl]amino-(2S)-2-hydroxy-4-phenylbutanoic acid as colorless needles. m.p.152°-154° C. Mass spectrometry: m/e 710 (M+1).

This N-protected product (100 mg) was subjected to hydrogenolysis in thesame manner as in the step (b) of Example 12 for the deprotection togive 67 mg of a crude powder of the titled compound. Recrystallizationfrom methanol-ethyl acetate gave 42 mg of colorless needles. m.p.217°-218° C. [α]_(D) ²⁵ +17.0° (c 1.0, 1 N-HCl). Mass spectrometry: m/e486 (M+1).

Elemental analysis--Found: C, 64.20, H, 7.33, N, 8.51%. Calcd. for C₂₆H₃₅ N₃ O₆ (molecular weight 485.64): C, 64.30; H, 7.28; N, 8.66%.

The following Examples 17-19 illustrate the formulation of thepharmaceutical composition of this invention.

EXAMPLE 17

(2S,3R)-Amino-2-hydroxy-4-phenylbutanoic acid i.e., (2S,3R)-AHPA (0.1 g)and mannitol (5 g) were taken up into distilled water to a total volumeof 1000 ml, and the solution obtained was sterillized in a conventionalmanner. The sterillized solution in 2 ml-portions was placed in viralesof glass and then freeze-dried. For use, the lyophilized product wasdissolved in sterile distilled water to make up an injectable solution.

EXAMPLE 18

One part by weight of (2S,3R)-AHPA was well mixed with 2000 parts byweight of lactose, and the mixture was passed through a 50 mesh screento give a powdery formulation.

EXAMPLE 19

One part by weight of (2S,3R)-AHPA, 770 parts of lactose, 200 parts ofcorn starch and 30 parts of polyvinylpyrrolidone (as binder) were wellmixed together. The mixture was wetted with a volume of ethanol andgranulated. The granules were mixed with 0.5 parts of finely dividedmagnesium stearate as the lubricant and compressed into the form oftablets each weighing 1 mg.

What we claim is:
 1. A new compound of the general formula ##STR28##wherein R₁ is a hydrogen atom or a hydroxyl group, R₂ is a hydrogen atomor an alkanoyl group of 2-7 carbon atoms or an alkoxycarbonyl group of2-7 carbon atoms, and R₃ is selected from the groups: ##STR29## where R₄and R₅ may be the same or different from each other and each is an alkylgroup of 1-7 carbon atoms, a hydroxyalkyl group of 1-7 carbon atoms, amercaptoalkyl group of 1-7 carbon atoms, a carboxamidoalkyl group of 2-8carbon atoms, an aminoalkyl group of 1-7 carbon atoms, aguanidyl-N-alkyl group of 1-7 carbon atoms, an alkylmercaptoalkyl groupof 2-7 carbon atoms, a carboxyalkyl group of 2-8 carbon atoms, an arylgroup, particularly phenyl, an aralkyl group of 7-8 carbon atoms or asubstituted aralkyl group of 7-8 carbon atoms, n is zero or 1, and theasterisk (*) denotes the R-configuration or S-configuration or acombination thereof.
 2. A new compound of the formula ##STR30## whereinR₁ is a hydrogen atom or a hydroxyl group, R₂ is a hydrogen atom or analkanoyl group of 2 to 7 carbon atoms or an alkoxycarbonyl group of 2 to7 carbon atoms, and R₆ is the group: ##STR31## wherein R₄ and R₅ are asdefined in claim
 1. 3. A new compound of the formula ##STR32## whereinR₁ is a hydrogen atom or a hydroxyl group, R₂ is a hydrogen atom or analkanoyl group of 2 to 7 carbon atoms or an alkoxycarbonyl group of 2 to7 carbon atoms, and R₇ and R₈ may be the same or different from eachother and each is an alkyl group of 1 to 7 carbon atoms, a hydroxyalkylgroup of 1 to 7 carbon atoms, a mercaptoalkyl group of 1 to 7 carbonatoms, an aminoalkyl group of 1 to 7 carbon atoms, a guanidyl-N-alkylgroup of 1 to 7 carbon atoms, an alkylmercaptoalkyl group, acarboxyalkyl group of 2 to 8 carbon atoms, a carboxamidoalkyl group of 2to 8 carbon atoms, an aryl group, particularly phenyl group, an aralkylgroup, particularly a phenyl (C₁ -C₄) alkyl group, especially benzyl, ora substituted aralkyl group, and n is 0 or 1, and the asterisk (*) eachdenotes the R-configuration or S-configuration.
 4. A new compound of theformula ##STR33## wherein A is a hydrogen atom or an alkanoyl group of 2to 5 carbon atoms, especially acetyl and B is the group: ##STR34## 5.The compound of claim 2 which is selectedfrom:((2S)-2-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol,(2S)-2-N-[(2'S,3'R)-3'-N-acetylamino-2'-hydroxy-4'-phenylbutanoyl]amino-4-methyl-pentanol,and(2S)-2-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl-(S)-leucyl]amino-4-methyl-pentanol.6. The compound of claim 3 which is selectedfrom:(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-leucine,(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-arginine,(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(R)-leucine,(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-glutamic acid,and(3R)-N-[(2'S,3'R)-3'-amino-2'-hydroxy-4'-phenylbutanoyl-(S)-leucyl]amino-(2S)-2-hydroxy-4-phenylbutanoicacid.
 7. A pharmaceutical immunopotentiating composition, comprising asactive ingredient the compound of the formula (VIII) ##STR35## whereinR₁ is a hydrogen atom or a hydroxyl group, R₂ is a hydrogen atom, analkanoyl group of 2 to 7 carbon atoms or an alkoxycarbonyl group of 2 to7 carbon atoms or an aralkyloxycarbonyl group of 7 to 8 carbon atoms,especially benzyloxycarbonyl, and D is the group: ##STR36## wherein R₄and R₅ may be the same or different from each other and each are analkyl group of 1 to 7 carbon atoms, a hydroxyalkyl group of 1 to 7carbon atoms, a mercaptoalkyl group of 1 to 7 carbon atoms, acarboxamidoalkyl group of 2 to 8 carbon atoms, an aminoalkyl group of 1to 7 carbon atoms, a guanidyl-N-alkyl group of 2 to 7 carbon atoms, analkylmercaptoalkyl group of 2 to 7 carbon atoms, a carboxyalkyl group of2 to 8 carbon atoms, an aryl group, particularly phenyl, an aralkylgroup of 7 to 8 carbon atoms, particularly benzyl, or a substitutedaralkyl group of 2 to 8 carbon atoms, n is zero or 1, and the asterisk(*) denotes the R-configuration or S-configuration, in combination witha pharmaceutically acceptable carrier for the active ingredient.
 8. Thepharmaceutical composition of claim 7, comprising as active ingredient acompound of the formula (VIIIa) ##STR37## wherein R₁ is a hydrogen atomor a hydroxyl group, A is a hydrogen atom or an alkanoyl group of 2 to 5carbon atoms, especially acetyl, and D' is the group: ##STR38## incombination with a pharmaceutically acceptable carrier for the activeingredient.
 9. The pharmaceutical composition of claim 7, comprising asactive ingredient the new compound of the formula (III) ##STR39##wherein R₁ is a hydrogen atom or a hydroxyl group, R₂ is a hydrogen atomor an alkanoyl group of 2-7 carbon atoms or an alkoxycarbonyl group of2-7 carbon atoms, and R₃ is selected from the groups: ##STR40## where R₄and R₅ may be the same or different from each other and each is an alkylgroup of 1-7 carbon atoms, a hydroxyalkyl group of 1-7 carbon atoms, amercaptoalkyl group of 1-7 carbon atoms, a carboxamidoalkyl group of 2-8carbon atoms, an aminoalkyl group of 1-7 carbon atoms, aguanidyl-N-alkyl group of 1-7 carbon atoms, an alkylmercaptoalkyl groupof 2-7 carbon atoms, a carboxyalkyl group of 2-8 carbon atoms, an arylgroup, particularly phenyl, an aralkyl group of 7-8 carbon atoms or asubstituted aralkyl group of 7-8 carbon atoms, n is zero or 1, and theasterisk (*) denotes the R-configuration or S-configuration.
 10. Aprocess of potentiating the immune response in a living animal includingman, which comprises administering orally or parenterally orintrarectally into the animal an immunopotentiatingly effective amountof the compound of the formula (VIII) ##STR41## wherein R₁ is a hydrogenatom or a hydroxyl group, R₂ is a hydrogen atom, an alkanoyl group of 2to 7 carbon atoms or an alkoxycarbonyl group of 2 to 7 carbon atoms oran aralkyloxycarbonyl group of 7 to 8 carbon atoms, especiallybenzyloxycarbonyl, and D is the group: ##STR42## wherein R₄ and R₅ maybe the same or different from each other and each are an alkyl group of1 to 7 carbon atoms, a hydroxyalkyl group of 1 to 7 carbon atoms, amercaptoalkyl group of 1 to 7 carbon atoms, a carboxamidoalkyl group of2 to 8 carbon atoms, an aminoalkyl group of 1 to 7 carbon atoms, aguanidyl-N-alkyl group of 2 to 7 carbon atoms, an alkylmercaptoalkylgroup of 2 to 7 carbon atoms, a carboxyalkyl group of 2 to 8 carbonatoms, an aryl group, particularly phenyl, an aralkyl group of 7 to 8carbon atoms, particularly benzyl, or a substituted aralkyl group of 2to 8 carbon atoms, n is zero or 1, and the asterisk (*) denotes theR-configuration or S-configuration, in combination with apharmaceutically acceptable carrier for the active ingredient, or of thecompound of the formula (VIIIa): ##STR43## wherein R₁ is a hydrogen atomor a hydroxyl group, A is a hydrogen atom or an alkanoyl group of 2 to 5carbon atoms, especially acetyl, and D' is the group: ##STR44## incombination with a pharmaceutically acceptable carrier for the activeingredient.
 11. (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-leucine.
 12. (2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-(S)-leucyl-(S)-arginine.